1. Field of the Invention
The present invention relates to a method for cultivation of filamentous fungi, and more particularly to a method for cultivation of filamentous fungi by using suspended nutritionally solid substrates.
2. Description of the Related Arts
Generally, the method for fermentation of filamentous fungi can include the use of solid-state culture or submerged culture. The conventional method for the most part uses solid-state culture. One example of producing metabolites from Monascus species is directly inoculating Monascus species into solid nutritional medium such as grain medium, for example rice or wheat. Then, fermentation is carried out. The mycelia of Monascus species penetrate into the surface of the solid nutritional medium and grow during the period of fermentation. The pigments produced are absorbed so that the color of the medium turns purple. In addition, the mycelia also penetrate inside the grain particles. This phenomenon facilitates the production of pigments. In 1991, Johns and Stuar employed Carrageenan particles consisting of synthetic medium to simulate grain in culture (Johns and Stuar, (1991) J. Industrial Microbiology, 8:23-28). However, due to reasons such as structure, nutrition components, etc, the synthetic medium is not suitable for Monascus species in the production based on solid-state culture. Generally, to adequately make use of medium and maintain optimal temperature and humidity, the operation procedures of conventional solid-state culture are complex and time-consuming. Moreover, some problems such as the restriction of oxygen transfer, contamination, etc., also make the scale-up difficult.
Furthermore, because of the disadvantages of solid-state culture described above, the quality and quantity of the products are usually unstable. To solve this problem, many studies have developed submerged culture to carry out the fermentation. In 1982, Lin and Lizuka employed saccharide and cereal meals as a carbon source to solve the problems of scale-up and operation control (Lin and Lizuka, (1982) Appl. Environ. Microbiology, 43:671-676). However, the yield of the pigments is significantly reduced using this method. In 1984, Evans and Wang employed algin (an inert substance in which no nutrient is contained) to immobilize Monascus species and then carry out submerged culture. The result showed the phenomenon of mycelia attachment conduces the production of the pigments (Evans and Wang, (1984) Applied and Environmental Microbiology 47(6): 1323-1326). In 1990, Mak et. al. introduced roller bottle culture, which has the properties of high oxygen mass transfer and low shear stress to provide an environment for mycelia attachment, and used glucose as a carbon source to carry out the cultivation of Monascus species (Mak et. al., (1990) Enzyme Microbiol. Technol., 12:965-968). However, due to the nature of the roller bottle, the scale of the production is too small using this method. In 1995, Lee et. al. used topioca starch as a carbon source and pointed out that a high initial starch concentration in the medium is not suitable for practical use due to high viscosity of the medium (Lee et. al., (1995) J. Fermentation of Bioengineering, 79(5): 516-518). They proposed a solid-liquid state culture with gelatinized starch block provided at the bottom and a dilute soluble starch at the upper part of the reactor. The cultivation was carried out in a stirred-tank bioreactor. However, their proposed approach might have scale-up problem since the interface area to volume ratio is small for a large-scale system.
These examples of submerged culture of filamentous fungi use nutritional medium such as algin and tapioca starch. All show poor fermentation yields.
Thus, the primary object of the present invention aims to improve the poor fermentation yields described above.
In addition, another object of the present invention is to carry out the submerged culture under the conditions of mycelia attachment and to avoid destroying mycelia and the attached material by the shear stress of the bioreactor, thus improving the process of producing filamentous fungi and to scale up the production of metabolites.
It is therefore an object of the present invention to provide a method for cultivation of filamentous fungi such as Monascus species by using a suspended nutritionally solid substrate, the method comprising the steps of: (a) preparing a medium comprising the nutritionally solid substrate; and (b) inoculating an inoculum into the medium comprising the nutritionally solid substrate in a bioreactor to carry out fermentation.
The method of the present invention can alternatively comprise a step of inoculating the filamentous fungi after step (a) to obtain an inoculum, then inoculating said inoculum into the medium comprising the nutritionally solid substrate in a bioreactor to carry out fermentation.
In the method according to the present invention, the bioreactor is a pneumatic bioreactor, more preferably an air-lift bioreactor with a net draft tube.
The method of the present invention can further comprise a step of cultivating the filamentous fungi using the fed-batch process, wherein the medium of the batch comprises a nitrogen source and the nutritionally solid substrate.